Process for producing antibiotic pa-180



United States Patent 3 Claims. (Cl. 19580) This invention relates to a new and useful plant inhibitory containing antibiotic called PA-l80. More particularly, it is concerned with its production by fermentation and with methods for its recovery and concentration from crude solutions. The invention includes within its scope dilute forms of 'PA-180, as well as crude concentrates and its purified forms. This novel product is valuable as a reversible plant growth inhibitor which preferentially inhibits root formation and as an antibiotic.

In the past, various chemicals and antibiotics have been foundto inhibit the growth of plants. These compounds reduce the overall plant height and frequently, reduce the plants growth rate, consequently, delaying flowering and prolonging the life span of the plant. Normally the Patented August 23, 1966 has been deposited with the American Type Culture Collection of Washington, DC, and given the number ATCC 14443. The cultural characteristics of this microorganism were determined by planting a culture thereof on media normally used for the identification of stretomycetes and observing the growth and other changes incident thereto.

This culture was identified by Dr. J. B. Routien as Streptomyces fradiae because it so closely resembled two authentic strains (W 3535 and NRRL 1195) of Streptomyces fmdiae that were simultaneously compared with it. All three cultures had the same lavender-brown aerial mycelium, the same types of spore-chains and the same general color of vegetative mycelium.

Our strain differed from the other two in not liquefying gelatin, not hydrolyzing starch and in producing a dark pigment in milk. However, strains of S. fmdiae are known that differ from the published description of this species (see Waksmans The Actinomycetes, Volume II, 1961, p. 212), so the differences listed here are not critical.

The cultural characteristics of ATCC 14443 are set forth in Table I, wherein the results are based upon six tubes or plates of each medium incubated for two weeks.

TABLE I Medium Amount of Aerial Mycelium and Sporulation Soluble Remarks Growth Pigment Glucose-asparagine agar Good Moderate amount; cream colored Lacking Reverse light yellow. Synthetic agar Moderate Good production; pinkish d Reverse pale orange-cream. Spore chains lavender. scattlered; hooks, loops and primitive I s ira s. Nutrient Agar Fair Lackin Slight brown Reverse brown. Glucose Agar Good o Lacking Reverse tan-cream. Milk do Scant; whitish Gray-br0wn Coagulation and peptonization in some tubes, no change in others; pH changed from 6.5 to 7.1. Cellnlnco N0 growth Potato Plug Fair Lat-kin Lackin Vegetative mycelium tan to dark cream. Dextrose Nitrate Broth Good Fair; white d0 Nitrites not detected, nitrates still present.

elatin do Good; grayish-white Olive No liquefaction. Starch Plates do Good; whit Lacking Reverse white to pale yellow; no hydrolysis. Calcium Malate Plates Excellent Excellent; becoming lavenderd0 Reverse pale yellow; malate digested. brown. Spore chains in hooks, loops; spores oval to oblong, 0.9 to 1.5g. Peptone Iron Agar.

N 0 H28 produced.

younger the plant, the more influence a retardant compound will exert on the overall height and rate of development of a plant. The advantages and uses of these inhibitory compounds are: improving the form and attractiveness of many kinds of ornamental flowers; shortening the stem height of crop plants that are subject to wind damage; decreasing the stem height of certain crop plants to facilitate harvesting; to retard development, to extend the harvest of crop plants over a longer period and to decrease the size of certain ornamental trees, thus, decreasing maintenance costs. The production by microorganisms of substances inhibitory to the growth of higher plants is Well documented. Curtis in Plant Physiology, 32, 56 (1957) reported two translocatable plant growth inhibitors which are produced by fungi. Brian in Symp. Soc. Exp. Biol, 11, 166 (1957) discussed four antibiotics which are inhibitory to root growth; two produced by actinomycetes (actidione and azaserine), one produced by a fungus (alternaric acid), and one produced by a bacterium (polymyxin).

production.

The new plant inhibitory containing fermentation antibiotic is formed during the cultivation under controlled 70 conditions of a microorganism which is a member of the genus Streptomyces. A living culture of this organism It is to be understood that for the production of the plant inhibitory containing PA-l80 the present invention is not limited to the aforementioned organism or to organisms fully answering the above description which is given only for illustrative purposes. ATCC 14443 is a species which was isolated from the soil and when the term Streptomyces fmdiae, ATCC 14443, is used, it is expressly-desired and intended to include the use of mutants produced from the described organism by various means such as X-radiation, ultraviolet radiation, nitrogen mustards and the like; and also to include microorganisms of this same new species regardless of their origin.

The invention includes within its scope processes for growing the microorganism Strepto myces fradiae ATCC 14443. The cultivation of the microorganism preferably takes place in an aqueous nutrient medium at a temperature of about 25 C. to 35 C. and under submerged conditions of agitation and aeration. which are useful for these processes include a carbohy- Nutrient media drate, such as sugars, starch, glycerol, corn meal; and a source of organic nitrogen, such as, for example, casein, soy [bean meal, peanut meal, wheat gluten, cottonseed meal, .lactalbumin and enzymatic digest of casein and tryptone. A source of growth such as distillers solubles, yeast extract, molasses fermentation residues as well as mineral salts such as sodium chloride, potassium phosphate, sodium nitrate, magnesium sulfate and trace minerals such as copper, zinc and iron may also be utilized with desirable results. If excessive foaming is encountered during fermentation, anti-foaming agents such as vegetable oils may be added to the fermentation medium. A

buffering agent such as calcium carbonate may also be added to the medium.

Inoculum for the preparation of PA180 by the growth of ATCC 14443 may be obtained by employing growth from slants on such media as Emersons agar or beef lactose. The growth may be used to inoculate either shake flasks or inoculum tanks for submerged growth or alternatively the inoculum tanks may be seeded from the shake flasks. The growth of the microorganism usually reaches its maximum in about two to four days. However, variation in the equipment used, the rate of aeration, the rate of stirring, etc. may affect the speed with which maximum activity is reached. In general, the fermentation is continued until a substantial amount of PA-l8-0 is produced. A period of about twenty-four hours to six days is sufi'icient for most purposes. Aeration of the medium in tanks for submerged growth is maintained at the rate of about one-half to two volumes of free air per volume of broth per minute. Aeration may be maintained by suitable types of agitators generally familiar to those in the fermentation industry. Aseptic conditions must, of course, be maintained throughout the transfer of the inoculum and throughout the growth of the microorganism.

After growth of the microorganism, the mycelium may be removed from the fermentation broth by means of standard equipment, such as filter-presses, centrifuges,v

and so forth. Thereafter, PA-180 may be recovered from the fermentation broth by several different procedures. Alternatively, the whole broth may be used as is or it may be dried. The PA-180 may be extracted from the aqueous fermentation broth at alkaline pHs preferably between about 6 and 10, by means of a variety of water immiscible organic solvents, including aromatic hydrocarbons, esters, ketones, lower alcohols and halogenated hydroearibons. Examples of these are diethyl ether, benzene, ethyl acetate, butyl acetate, methyl isobutyl ketone, butanol and chloroform. Subsequently, PA- 180 is recovered by evaporating the water immiscible solvent to dryness. Further methods of recovery which suggest themselves include absorption on charcoal with subsequent elution and development on alumina columns.

The product of the aforementioned fermentation process is PA-180 which is a light tan solid. Papergram studies of this material utilizing various chemical and physical detection techniques disclosed that it is heterogeneous in nature and bioautographs showed the presence of several dominant antibiotic substances. PA-l 80 is non toxic when administered to mice at a 200 mg./ kg. dosage and its antibiotic activity is readily demonstrated against a variety of microorganisms. More particularly this activity has been determined for PA180, and for the Water soluble and ethanol soluble fractions thereof. This data is presented in Tables II, III, and IV, wherein the columns entitled meg/ml. are the minimum inhibitory activities of PA-180, and the isolated fractions thereof.

TABLE II Organism: PA-l80 meg/ml. M icrococcus pyogenes var. aureus 25 Streptococcus pyogenes 6.25 Streptococcus faecalis 3.12 Erysz'pelothrux rhusiopathiae 100 Diplococcus pneumoniae 100 Corynebacterium diphtheriae 6.25 Listeria monocytogenes 100 Bacillus subtilis 12.5 Clostridium perfringens 100 Lactobacillus casei 100 Bacterium ammaniagenes 100 Aerobacter aerogenes 100 Escherichia coli 100 Proteus vulgaris 100 Pseudomonas aeruginosa 100 Salmonella typhosa 100 4 Organism: PA-l meg/ml. Salmonella pullorum Salmonella gallinarum 100 Klebsiella pneumoniae 100 Nesseria gonnorheae 100 Hemophilus influenzae 100 Shigella sonnei 100 Erwinia omylovora 100 Phytomonas tumefaciens 100 Brucclla bronchz'septica 100 Malleomyces mallei 100 Desulfovibrio desulfuricans 100 Vibrio comma 100 pi Posteurella multocido 100 Candida albicons 100 Saccharomyces cerevisiae 6.25 Micrococcus pyogenes var. aureus 376 25 M icrococcus pyogenes var. aureus 400 50 Mycobactcrz'um 607 50 Mycobacterium berolinense 12.5 Pityrosporum ovale 12078 200 Pityrosporium ovale Traub 200 TABLE III Water soluble fraction Organism: of PA- (meg/ml.) Staphylococcus aureus 50 Staphylococcus aureus 400 100 Streptococcus pyogenes 12.5 Streptococcus pyogenes 98 5O Streptococcus faecalis 100 Erysipelothrix rhusz'opathiae 12.5 Aerobacter aerogenes 2S Escherichia coli 100 Proteus vulgaris 25 Pseudomonas aeruginosa 100 Salmonella typhosa 50 Klebsiellol pneumoniae 12.5 Hemophilus pertussis 1.56 Klebsiella pneumoniae 50 TABLE IV Ethanol soluble fraction Organism: of PA-180 (meg/ml.) Staphylococcus aureus 6.25 Staphylococcus aureus 400 100 Streptococcus pyogenes 6.25 Streptococcus pyogenes 98 6.25 Streptococcus fuecalis 25 Erysipclothrix rhusiopathiae 6.25 Aerobacter aerogenes 100 Escherichia coli 100 Proteus vulgaris 100 Pseudomonas aerugz'nosa 100 Salmonella typhosa 100 Klebsiella pneumoniae 25 Hemophilus pertussis 25 Klebsiella pneumoniae 50 The plant inhibitory factor may be a small but potent fraction of PA-180. When plants are treated with PA- 180, their growth is remarkably inhibited with no apparent toxic effects. This effect is reversible, that is, if the dwarfed plants are no longer treated with this material they immediately resumetheir normal growth. The effect of this new inhibitor was tested by the duckweed growth test which is widely employed in the evaluation of herbicides and growth inhibitors. This test is described in the Journal of Agricultural and Food Chemistry, volume 2, pages 178-182 (1954). The usual procedure employed in the duckweed test is to add the subject compound or mixtures to a synthetic nutrient solution which has been sterilized by filtration through a sintered glass funnel and to observe the growth of the duckweed (Lemna minor) over a period of time at various concentrations of the subject compounds or mixture. The plant nutrient media usually contain the cations, potassium, magnesium, calcium and the anions, sulfate, nitrate and phosphate. A detailed description of this type of nutrient media is found in the text, Plant Science Formula, McLean and Cook (Macmillan), 1950, second edition.

solid carrier so that the mixture is in the form of powder or dust. This term is also meant to encompass mixtures which are suitable for use as sprays including solutions, suspensions or emulsions of the agent of this invention in 5 a liquid propellant which boils below room temperature, The following table lists the inhibitory eflect of various at ordinary pressures. concentrations of PA-180, along with similar data for The term agricultural carrier includes any and all other microbial metabolic products including antibiotics, of those agriculturally acceptable agents in which the some well-known plant growth inhibitors and some genproduct of this invention is dispersed. It includes, thereeral metabolic inhibitors, on the growth of Lemna minor 10 fore, the solvents of a true solution, the liquid phase of when studied by the duckweed test. The results are exsuspensions, emulsions or sprays, the semi-solid carriers pressed as percent change of the controls on a wet weight and the solid phase of dust and powders. basis. It was also noted that although PA-l80 greatly The are many solvents which can be utilized for the inhibited the growth of Lemna minor, it did not exhibit preparation of solutions, suspensions or emulsions of the any phytotoxicity. material of this invention. Although any non-toxic liquid TABLE V Percent Change from Control (wet weight basis) Compound Comments 1p.p m. 5p.p.m. l0p.p.m. p.p.m.

-15--60 -85 -90 -95 Tiny plants at 5-20 p.p.m.

Dead Dead Dead Dead Anisomycl'n -80 -90 -90 Dead Leaves small, chlorotic; complete root inhibition. Azaserine Dead Dead Dead Dead Bacitracin 0 +80 +100 +130 Catenulin Dead Dead Dead Dead' Duramycin..- +35 +35 +30 Root inhibition at 20 p.p.m. Griseofulvim. -10 -15 20 Kojic Acid +40 +70 +65 +70 Oleandornycin PO, 0 0 0 0 Penicillin 6%.. +60 +225v +230 +220 Penicillin s +100 +210 +290 +240 Polymyxin B -15 -15 Dead Dead Triehomycin 0 +5 +10 +35 Azauracil -20 Dead Dead Dead 3-anlino-l,2,4-triazole -5 -10 -70 Clsilggosis and root inhibition at p.p,ln. (l-hydroxy-fi-isopropyl-Z- +10 +20 +20 +25 methyl-phenyl)-trimethyl ammonium chloride, 1- piperidine-carboxylate. (4-hydroxy-5-isopropyl-2- +15 +15 +10 +10 methyl-phenyl)-trimetl1yl ammonium iodide, 1- piperidine-carboxylate. 2,4-dinitr0phenol -25 Dead Dead Dead (2-bron1oethyl)trimethyl- 0 +10 +5 ammonium bromide. (2-chloroethyl)trimethyl- +55 +20 0 0 ammonium chloride. (2,3n-propylene)-trimethy1- +40 +25 10 0 ammonium chloride. Maleic hydrazide 0 -10 -25 -40 Root inhibition at 10-20 ppm.

The plant inhibitory portion of PA-180 is so active that 50 a concentration level from about 0.10 part per million may be employed, the preferred solvent is water. Howto about 1000 parts per million of the fermentation prodever, for certain applications it may be advantageous to not PA-18O will produce an unusually high degree of resort to mixtures of solvents. If the active agent is to inhibition. Although some effect may be obtained by be applied as a spray, it is convenient to dissolve it in a using less than 0.10 part per million, the growth in- 59 suitable solvent and to disperse the resulting solution in hibitory effect may be variable audit is advisable not to a liquid propellant which boils below room temperature. use appreciably smaller amounts. The preferred amounts For such applications it is a better to employ a true soluare levels in the range of about 1.0 part per million to tion of the active agent, although it is possible to employ about 100 parts per million, although this will vary somesuspensions or emulsions. what with the particular plant being treated. 60 For use as a powder or dust the active ingredient of The material of this invention, PA-18-0 may be emthis invention is mixed with any number of extending ployed alone or in combination with other plant growth agents either organic or inorganic in nature which are regulatory ingredients. When the material of this insuitable for the manufacture of an agricultural pulverulent vention is employed, it is most economical to use it in a preparation. Such extending agents include for example, dispersed form in a suitable agricultural carrier. 63 urea, potting soil, inorganic mineral salts and others. When it is said that this material is dispersed, it means These mixtures may be used in the dry form or, by the that the particles may be molecular in size and held in addition of water the dry powder can be dispersed as true solution in a siutable solvent or that the particles may a solution or suspension suitable for use as sprays. be colloidal in size and dispersed through a liquid phase F In all of the forms described above, the dispersions in the form of a suspension or an emulsion. It also int 0 may be provided ready for use or they may be provided eludes particles which are dispersed in asemi-solid viscous in a concentrated form suitable for mixing with other carrier in which they may be actually dissolved or held extending agents before use. in suspension with the aid of a suitable emulsifying or The following examples are given solely for the purwetting agent. The term dispersed also means that the pose of illustration only and are not to be construed as particles may be mixed with and spread throughout a limitation of this invention, many variations of which 3,2es,4.1s

are possible without departing from the spirit or scope thereof.

Example I A slant of Streptomyces fradiae ATCC 14443 on Emerson agar was cultivated under controlled conditions to develop spores for the purpose of inoculating a nutrient medium of the following compositions:

Grams Cerelose (dextrose hydrate) 10 Soybean meal 10 Sodium chloride Distillers solubles 5 Calcium carbonate 1 following composition:

Grams/liter Cerelose Sodium chloride 5 Curbay B-G (distillers molasses solubles) 5 Corn starch 10 Soybean meal 10 This medium was adjusted to .pH 7 with potassium hydroxide, treated with 1 gram of calcium carbonate per liter, and sterilized in the usual manner, before transferring the broth and mycelium thereto. After seeding the medium with the organism from the shake flasks, the mixture was subjected to agitation and aeration under sterile conditions for three days. The mycelium was removed by filtration and the filtrate extracted twice with one-quarter volume of ethyl acetate. The ethyl acetate extract was concentrated in vacuo to one-twentieth its volume. This concentrate was washed twice with onetenth volume of 5% sodium bicarbonate, and then twice with one-tenth volume of water. The ethyl acetate extract was then distilled azeotropically to remove the water present, and the inhibitory containing product PA-180, was precipitated by the addition of five volumes of hexane. The precipitated powder was removed by filtration. This powder represented a lot) fold concentration over the original broth after freeze drying the same. The product thus prepared was fur ther purified by dissolving the same in methylene chloride and fractionally precipitating impurities with carbon tetrachloride, leaving the PA-18O in solution. Upon evaporation of the solvent, a tan colored solid was obtained.

Example 11' Another fermentation medium was prepared from the following materials 2 Distillers solubles 5 These materials were added to one liter of water and the pH of the resulting mixture was adjusted to between 7 and 7.2 with potassium hydroxide. Five grams of calcium carbonate were added to act as a buffer during the fermentation. The medium was then autoclaved and seeded under sterile conditions with Strep-tomyces fradia'e ATCC 14443 inoculum prepared in accordance with the procedure set forth in Example I. After subjecting the inoculated medium to aeration and agitation under sterile conditions at about 35 C. for 2 days, the filtered broth was found to contain a very high concentration of PA-l80.

Exa'mple III To a mixture of grams of pulverized calcium carhon-ate, 2 grams of olein and one gram of slaked lime, there was added sufiicient PA- to give a mixture containing 100 parts per million of PA180 and the mixture was ground in a ball mill. The resulting powder is easily scattered, has good adhesive properties and promotes growth when applied to plants.

Example IV A mixture of 5 milligrams of PA-ISO, 25 grams of talcum, 4 grams of sodium dibutyl naphthalene sulfonate, 4 grams of casein and 5 grams of sodium carbonate was ground in a ball mill. The mixture was added to an equal quantity of ground calcium carbonate and thor oughly mixed. This powder may be suspended in water immediately before use and gives a growth-promoting suspension which is sufliciently stable for use in spraying applications.

Example V A mixture is prepared containing 10 milligrams of PA-180, 20 grams of xylene and 80 grams of Turkey red oil. This solution can be readily emulsified in water and the emulsion is suitable as a spray.

Exa'mple VI An aqueous solution is prepared containing 0.005% of PA-l80, 10% men, 5% of phosphoric acid and 5% of potassium hydroxide. This solution is diluted with water immediately before use and gives a growth promotion solution suitable for application to plants.

What is claimed is:

1. A process for producing antibiotic PA180 which comprises cultivating Streptomyces fradiae ATCC 14443 in an aqueous nutrient medium under submerged aerobic conditions until substantial activity is imparted to said medium.

2. A process as claimed in claim 1 wherein the product is recovered from the fermentation broth.

3. A process as claimed in claim 1 wherein the growth of the microorganism is conducted at a temperature between about 25 C. and about 35 C. for about two to four days.

References Cited by the Examiner Pfizer, Handbook of Microbial Metabolites (1961), pp. 41, 42, 128, 140, 386388.

JULIAN S. LEVITT, Primary Examiner.

S. ROSEN, Assistant Examiner. 

1. A PROCESS FOR PRODUCING ANTIBIOTIC PA-180 WHICH COMPRISES CULTIVATING STREPTOMYCES FRADIAE ATCC 14443 IN AN AQUEOUS NUTRIENT MEDIUM UNDER SUBMERGED AEROBIC CONDITIONS UNTIL SUBSTANTIAL ACTIVITY IS IMPARTED TO SAID MEDIUM. 